Structural and Biochemical Characterization of SrcA, a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

Abstract

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

Identification of the Docking Site between a Type III Secretion System ATPase and a Chaperone for Effector Cargo

“…predicted to be the T3SS-2 ATPase based upon its similarity to other characterized T3SS ATPases and upon its enzymatic activity that has been demonstrated in vitro (18,44). We first confirmed the essential activity of this enzyme for bacterial infection fitness by competing a ⌬ssaN mutant against wild-type bacteria in C57BL/6 mice.…”

“…Protein Purification, Activity Assays, and Circular DichroismSsaN protein was produced and purified as described previously (18). ATPase activity of purified SsaN ⌬1-89 variants was determined by measuring the release of total phosphate at 37°C in a microplate assay with BIOMOL Green according to the manufacturer's instructions (Enzo Life Sciences).…”

“…The second T3SS, encoded by SPI-2, translocates effector proteins into the host cell that are necessary for ensuring intracellular survival and replication (11,12). Chaperone-effector interactions have been charted extensively for both the T3SS-1 (13-16) and T3SS-2 (17)(18)(19)(20)(21)(22), and a number of functions attributed to secretion chaperones beyond that of effector binding have been demonstrated in Salmonella (23,24).…”

A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

“…Later on, it became evident that some T3SC bind to multiple effector proteins (reviewed in reference 13). These class IB or multiple-cargo chaperones have been identified and characterized from different bacteria, including pathogenic Escherichia coli (CesT), Shigella (Spa15), Salmonella (InvB and SrcA), Chlamydia (McsC), and Xanthomonas (HpaB) (4,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Given that these multiple-cargo chaperones contribute to the biological function of various effectors, it is likely that they play a significant role during infection.…”

Impact of the N-Terminal Secretor Domain on YopD Translocator Function in Yersinia pseudotuberculosis Type III Secretion

“…Within this platform exists a conserved ATPase present in all T3SSs that is responsible for energizing substrate secretion (68,70). Ample biochemical evidence indicates that effectors can interact with their cognate ATPases either directly or indirectly via their T3S chaperones (2,10,15,33,50,68,70,73,74). However, there is still no published report demonstrating binding between the ATPase and translocatorchaperone complexes.…”